Our aim is to understand in atomic detail the natural engineering behind the central multifaceted role played by ubiquinone-10 in biological energy conversion, namely the chemical mechanisms that lead up to and drive the formation of electric potentials and proton gradients across membranes. We focus on the ubihydroquinone-cytochrome c oxidoreductase (bc1 complex) that is at the core of virtually all respiratory and photosynthetic electron transfer systems. We target the bc1 complex's distinct Qo and Qi sites that interface with the ubiquinone-10 membrane pool. The Qo site catalyses a remarkable, concerted two-electron ubiquinone oxidation-reduction that is central to the primary energy conversion reaction, while the Qi site plays an essential part of the secondary reactions in catalysing two, sequential one-electron transfers. Both sites are the locus of action of an array of site- specific inhibitors. Mechanistically, little is known about how the sites exploit the same ubiquinone molecule in such different ways or how different inhibitors impede catalysis. We intend to use exotic quinones, inhibitors and analogues and mutagenisis to identify the molecular factors that govern these Q-site affinities, specificities and pool exchange dynamics, following the pattern set by successful work on the Q-sites of the photosynthetic reaction center. Flash-activatable chromophores attached to selected positions on the bc1 complex will be used to dissect the energetic states and microscopic rates that lead up to and include Qo site catalysis. Integral to these processes is the [2Fe2S] cluster which through its ligand interactions with quinones and inhibitors profoundly influences the definition of the Qo site action. The nature of ligand interactions of the [2Fe2S] cluster will be described using model compounds, while the [2Fe2S] subunit's dynamic motion, emerging from the first crystal structures, will be probed by time-resolved fluorescence anisotropy, resonance energy transfer and single molecule polarization studies. In the longer term these results will guide us to explore the Q-sites and mode of energy conversion in the little understood respiratory NADH- ubiquinone oxidoreductase. Similar progress will stimulate the synthesis of simple four-helix bundle proteins designed to reflect engineering of sites of the Qo and Qi type. Chemical and physical understanding of how Q-sites function and fail will potentiate biomedical application a) by knowing the mode of action of natural inhibitors, drugs, and herbicides, and possibly of cellular regulators, b) by recognizing the mechanistic roots of potentially damaging respiratory byproducts (e.g. superoxide) that emanate from these sites; and c) by enhancing the use of quinones in metabolic therapy.